2.2 DNA extraction, RADseq library preparation and sequencing
Genomic DNA was extracted from each whole specimen using a DNeasy Blood
& Tissue Spin Column Kit (Qiagen, Venlo, Netherlands), including the
RNase treatment suggested by the manufacturer. The extraction was
performed in whole specimens previously washed with sterilised water
three times to remove any external contaminant in a room never exposed
before to medflies. The DNA was quantified using Qubit 2.0 dsDNA HS Kit
(Thermo Fisher Scientific, Waltham, US-MA) and sent to GenePool
laboratories (Edinburgh, Scotland) for RAD-tag library preparation and
sequencing. Briefly, DNA samples were digested
using SbfI high-fidelity restriction enzyme (NEB Inc., Ipswich,
US-MA). P1 adapters, each with a unique eight bp molecular identifying
sequence, were ligated using T4 DNA Ligase (NEB) to allow 16 individuals
per lane multiplexing. Fragments were pooled and size selected (300-700
bp) prior to purification with Qiagen columns. Fragment ends were
repaired using the Quick Blunting Kit (NEB), and P2 adapters were
ligated using T4 DNA Ligase. Minelute columns (Qiagen) were used to
purify DNA. PCR enrichment of the libraries was performed using Phusion
Flash High-Fidelity PCR Master Mix (NEB) and size selected using gel
electrophoresis. The sequencing of RAD tags was done on Illumina GAIIx
(Illumina Inc., San Diego, US-CA) following standard protocols, and data
is available via CSIRO Data Collection (Elfékih, Arias, & Vogler, 2020)